Class I-type lysyl-TRNA synthetase

ABSTRACT

A protein with canonical lysyl-tRNA synthetase activity was purified from  Methanococcus maripaludis , cloned, and sequenced. The predicted amino acid sequence of the enzyme indicated a novel class I polypeptide structurally unrelated to class II lysyl-tRNA synthetase reported in eubacteria, eukaryotes, and the Crenarchaeote  Sulfobus solfataricus . A similar class I polypeptide was isolated from  Borrelia burgdorferi , the causative agent of Lyme disease, and an open reading frame encoding a class I-type lysyl-tRNA synthetase was identified in the genome of  Treponema pallidum , the causative agent of syphilis. The  B. burdorferi  gene encoding tRNALysl was cloned and used to make tRNA in vitro. The fundamental difference between pathogen and host in an essential enzyme suggests that class I-type lysyl-tRNA synthetase provides a target for the development of medical and veterinary therapeutics and diagnostics for Borrelia and other microorganism infections.

RELATED APPLICATION DATA

This application is the U.S. National Stage of International Application No. PCT/US98/18968, filed on Sep. 9, 1998, which claims priority benefit of provisional U.S. application Ser. No. 60/058,420, filed on Sep. 10, 1997.

TECHNICAL FIELD OF THE INVENTION

This invention relates to a new class I-type lysyl-tRNA synthetase isolated from archaebacteria and Borrelia.

BACKGROUND OF THE INVENTION

Lysyl-tRNA synthetase (LysRS) is essential for the translation of lysine codons during protein synthesis. In spite of the necessity for this enzyme in all organisms and the high degree of conservation among aminoacyl-tRNA synthetases (1), genes encoding a LysRS homologue have not been found by sequence similarity searches in the genomes of two Archaea, Methanococcus jannaschii (2) and Methanobacterium thermoautotrophicum (3). This raises the possibility that. LysRS, like the asparaginyl- and glutaminyl-tRNA synthetases (4), is not present, with lysyl-tRNA (Lys-tRNA) synthesized by tRNA-dependent transformation of a misacylated tRNA (5). Alternatively, these organisms may contain a LysRS activity encoded by a gene sufficiently different to those previously identified to prevent its detection by sequence similarity searches.

SUMMARY OF THE INVENTION

This invention confirms the latter hypothesis and provides isolated and purified class I-type lysyl-tRNA synthetase (hereafter sometimes denoted herein as LysRSI) and active fragments and variants thereof, DNA and RNA sequences encoding class I-type lysyl-tRNA synthetase (and biological equivalents and fragments thereof), and methods for screening for class I-type lysyl-tRNA synthetase inhibitors for medical and veterinary use. It further provides methods for screening for infection of an organism by microorganisms expressing class I-type lysyl-tRNA synthetase.

DESCRIPTION OF THE FIGURES

FIG. 1 is a line graph providing data related to the direct attachment of lysine to tRNA by M. maripaludis protein extracts. Aminoacylation reactions were performed as described in the Examples that follow in the presence of 200 μg RNA-free total protein prepared from an S160 extract in a reaction volume of 130 μl. Aliquots (20 μl) were periodically removed and analyzed. Reactions were performed in the presence of: open circles, 20 μM (¹⁴C)lysine; closed circles, 20 μM (¹⁴C)lysine and the 19 canonical amino acids (800 μM (¹²C)) except for lysine; triangles, 20 μM (¹⁴C)lysine and 800 μM (¹²C)lysine.

FIG. 2 shows alignment of class I-type lysyl-tRNA synthetase amino acid sequences. The Figure uses the following abbreviations for the amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val, W, Trp; and Y, Tyr. The sequences were aligned using the CLUSTAL W program (20). The sequences shown are from Pyrococcus fuliosus (PF, SEQ ID NO: 1), Pyrococcus horikoshii (PH, SEQ ID NO: 2), Borrelia burgdorferi (BB, SEQ ID NO: 3), Treponema pallidum (TP, SEQ ID NO: 4), Methanococcus jannaschii (MJ, SEQ ID NO: 5), Methanococcus maripaludis (MM, SEQ ID NO: 6, expressed in Example 1 as a construct with an extra methionine at the end), Archaeoglobus fulgidus (AF, SEQ ID NO: 7), Methanobacterium thermoautotrophicum (MT, SEQ ID NO: 8) and Rhodobacter capsulatus (RC, SEQ ID NO: 9).

FIG. 3 is a line graph showing aminoacylation of M. maripaludis tRNA by purified M. maripaludis His₆-LysRS. Aminoacylation reactions were performed as described (20 μl samples) in the presence of 20 nM enzyme and the following amino acids: open circles, 20 μM (¹⁴C)lysine; closed circles, 20 μM (¹⁴C)lysine and the 19 canonical amino acids (800 μM (¹²C)) except for lysine; triangles, 20 μM (¹⁴C)lysine and 800 μM (¹²C)lysine. The squares represent an aminoacylation reaction performed with 20 μM (¹⁴C)lysine and 50 nM control protein preparation (His₆-glutamyl-tRNA reductase). The level of product formation at 30 minutes indicates that the M. Maripaludis total tRNA preparation contains approximately 32 pmol lysine tRNA per A₂₅₀ unit, in good agreement with the value for commercial E. coli total tRNA reagents (40 pmol/A₂₅₀, Boehringer Mannheim).

FIG. 4 is a line graph showing aminoacylation of E. coli tRNA by purified B. burgdorferi His₆-LysRS. Aminoacylation reactions were performed as described (20 μl samples) in the presence of 20 nM enzyme and the following amino acids: open circles, 20 μM (¹⁴C)lysine; closed circles, 20 μM (¹⁴C)lysine and the 19 canonical amino acids (800 μM (¹²C)) except for lysine; triangles, 20 μM (¹⁴C)lysine and 800 μM (¹²C)lysine.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based upon the isolation and sequencing of a eury-archaeal class I-type lysyl-tRNA synthetase distinguishable from class IIc-type lysyl-tRNA synthetase found in bacteria and eukarya, and the identification of genes for analogous sequences in other microorganisms, including microorganisms of medical and veterinary importance.

The sequencing of euryarchaeal genomes had suggested that the essential protein lysyl-tRNA synthetase (LysRS) is absent from such organisms. However, as summarized above and set out in the Examples that follow, a single 62-kD protein with canonical LysRS activity was purified from Methanococcus maripaludis, and the gene that encodes it, cloned. The predicted amino acid sequence of M. maripaludis LysRS is similar to open reading frames of unassigned function in both Methanobacterium thermoautotrophicum and Methanococcus jannaschii, but is unrelated to canonical LysRS proteins reported in eubacteria, eukaryotes, and the Crenarchaeote Sulfolobus solfataricus. The presence of amino acid motifs characteristic of the Rossmann dinucleotide binding domain identify M. maripaludis LysRS as a class I aminoacyl-tRNA synthetase, in contrast to the known examples of this enzyme, which are class II synthetases.

Analysis of the genomic sequences of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame with over 55% similarity at the amino acid level to archaeal class I-type lysyl-tRNA synthetase. In contrast, no coding region with significant similarity to any class-II type lysyl-tRNA synthetase could be detected. Heterologous expression of this open reading frame in Escherichia coli led to the production of a protein with canonical lysyl-tRNA synthetase activity in vitro. Analysis of B. burgdorferi mRNA showed that the lysyl-tRNA synthetase encoding gene is constitutively expressed in vivo. The data indicate that, like the archaeabacteria described above, B. burgdorferi also contains a functional class I-type lysyl-tRNA synthetase, again in contrast to most other bacteria and eukaryotes which contain class II-type enzymes.

The kinetic parameters of B. burgdorferi lysyl-tRNA synthetase were further assessed by amplifying and cloning the gene for B. burgdorferi tRNA-Lys1 and using the gene to make a tRNA in vitro using standard techniques. The rate of formation of Lys-tRNA was found to be the same with synthetic substrate as observed for other class I-type lysyl-tRNA synthetases with tRNAs synthesized in vivo.

This invention thus provides isolated and purified class I-type lysyl-tRNA synthetase (hereafter sometimes denoted herein as LysRSI) and active fragments and variants thereof, DNA and RNA sequences encoding class I-type lysyl-tRNA synthetase (and biologically equivalents and fragments thereof), and methods for screening for class I-type lysyl-tRNA synthetase inhibitors for medical and veterinary use. It further provides methods of detecting infection of an organism which expresses class II-type lysyl-tRNA synthetase by a microorganism expressing class I-type lysyl-tRNA synthetase.

In one embodiment, the invention provides purified and isolated DNA encoding the deduced LysRSI polypeptide set out as MM in FIG. 2 (SEQ ID NO: 6), degenerate and complimentary sequences to SEQ ID NO: 10, and sequences that hybridize under stringent conditions with the sequence. In another embodiment, the invention provides purified and isolated DNA encoding the deduced LysRSI polypeptide set out as BB in FIG. 2 (SEQ ID NO: 3), degenerate and complementary sequences to SEQ ID NO: 11, and sequences that hybridize under stringent conditions with the sequence.Also encompassed by this invention are cloned sequences defining LysRSI, which can then be used to transform or transfect a host cell for protein expression using standard means. Also encompassed by this invention are DNA sequences homologous or closely related to complementary DNA described herein, namely DNA sequences which hybridize to LysRSI cDNA, particularly under stringent conditions that result in pairing only between nucleic acid fragments that have a high frequency of complementary base sequences, and RNA corresponding thereto. In addition to the LysRSI-encoding sequences, DNA encompassed by this invention may contain additional sequences, depending upon vector construction sequences, that facilitate expression of the gene. Also encompassed are sequences encoding synthetic LysRSI peptides or polypeptides exhibiting enzymatic activity and structure similar to isolated or cloned LysRSI. These are referred to herein as “biological equivalents or variants,” and in some embodiments have at least about 50%, preferably at least about 60% to 85%, and more preferably, at least about 90% sequence homology with LysRSI.

Because of the degeneracy of the genetic code, a variety of codon change combinations can be selected to form DNA that encodes LysRSI of this invention, so that any nucleotide deletion(s), addition(s), or point mutation(s) that result in a DNA encoding the protein are encompassed by this invention. Since certain codons are more efficient for polypeptide expression in certain types of organisms, the selection of gene alterations to yield DNA material that codes for the protein of this invention are preferably those that yield the most efficient expression in the type of organism which is to serve as the host of the recombinant vector. Altered codon selection may also depend upon vector construction considerations.

DNA starting material which is employed to form DNA coding for LysRSI peptides or polypeptides of this invention may be natural, recombinant or synthetic. Thus, DNA starting material isolated from cultures of microorganisms, constructed from oligonucleotides using conventional methods, obtained commercially, or prepared by isolating RNA coding for LysRSI, and using this RNA to synthesize single-stranded cDNA which is used as a template to synthesize the corresponding double stranded DNA, can be employed to prepare DNA of this invention.

DNA encoding the peptides or polypeptides of this invention, or RNA corresponding thereto, are then inserted into a vector, and the recombinant vector used to transform a microbial host organism. Example host organisms useful in the invention include, but are not limited to, bacterial (e.g., E. coli or B. subtilis), yeast (e.g., S. cerevisiae), mammalian (e.g., mouse fibroblast or other cell line) or insect (e.g., baculovirus expression system) cells. This invention thus also provides novel, biologically functional viral and circular plasmid RNA and DNA vectors incorporating RNA and DNA sequences describing LysRSI or functional LysRSI fragments generated by standard means. Culture of host organisms stably transformed or transfected with such vectors under conditions facilitative of large scale expression of the exogenous, vector-borne DNA or RNA sequences and isolation of the desired polypeptides from the growth medium, cellular lysates, or. cellular membrane fractions yields the desired products.

The present invention thus provides for the total and/or partial manufacture of DNA sequences coding for LysRSI, and including such advantageous characteristics as incorporation of codons preferred for expression by selected non-mammalian hosts, provision of sites of cleavage by restriction endonuclease enzymes, and provision of additional initial, terminal or intermediate DNA sequences which facilitate construction of readily expressed vectors. Correspondingly, the present invention provides for manufacture (and development by site specific mutagenesis of cDNA and genomic DNA) of DNA sequences coding for microbial expression of LysRSI analogues which differ from the form specifically described herein in terms of identity or location of one or more amino acid residues (i.e., deletion analogues containing less than all of the residues specified for the protein, and/or substitution analogues wherein one or more residues are added to a terminal or a medial portion of the polypeptide), and which share or alter the biological properties of LysRSI described herein.

DNA (and RNA) sequences of this invention code for all sequences useful in securing expression in procaryotic or eucaryotic host cells of peptide or polypeptide products having at least a part of the primary structural conformation, and one or more of the biological properties of LysRSI which are comprehended by: (a) the DNA sequences encoding LysRSI as described herein, or complementary strands; (b) DNA sequences which hybridize (under hybridization conditions) to DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b) above. Specifically comprehended are genomic DNA sequences encoding allelic variant forms of LysRSI included there in, and sequences encoding RNA, fragments thereof, and analogues wherein RNA or DNA sequences may incorporate codons facilitating transcription or RNA replication of messenger RNA in non-vertebrate hosts.

The invention also provides the LysRSI peptides or polypeptides encoded by the above-described DNA and/or RNA, obtained by isolation or recombinant means, as well as the polypeptide set out as MM in FIG. 2 (SEQ ID NO: 6), BB in FIG. 2 (SEQ ID NO: 3), and their structural analogues. The invention correspondingly provides peptide mimics such as peptide fragments of LysRSI and functionally equivalent counterparts that demonstrate its enzymatic activity. For example, alterations in known sequences can be performed to enhance enzyme activity measurements or other properties desirable for use in screening methods and the like.

Isolation and purification of peptides and polypeptides provided by the invention are by conventional means including, for example, preparative chromatographic separations such as affinity, ion-exchange, exclusion, partition, liquid and/or gas-liquid chromatography; zone, paper, thin layer, cellulose acetate membrane, agar gel, starch gel, and/or acrylamide gel electrophoresis; immunological separations, including those using monoclonal, polyclonal, and/or fusion phage antibody preparations; and combinations of these with each other and with other separation techniques such as centrifugation and dialysis, and the like.

It is an advantage of the invention that the isolation and purification of LysRSI provides a polypeptide that is useful in screens for LysRSI inhibitors. LysRSI inhibitors are typically identified in screening assays when test compounds inhibit a functional property of the enzyme in an assay. By “LysRSI inhibitor” is meant any inhibitor of LysRSI function, antibodies to LysRSI or LysRSI functional fragments that inhibit enzymatic activity, receptor antagonists, and the like, and mixtures thereof. Inhibitors that do not inhibit class II-type lysyl-tRNA synthetase are particularly preferred. Any standard assay such as that described in the Examples that follow can be used to screen large collections of test compounds, or screen monoclonal, polyclonal, or fusion phage antibodies that inhibit enzymatic activity. Mixtures of inhibitors can also be employed. For example, the invention provides a method for screening for the presence or absence of class I-type lysyl-tRNA synthetase enzyme inhibition by a test sample comprising incubating the test sample with the enzyme and its substrates for a time under conditions sufficient to observe enzymatic activity in a control incubation of enzyme and substrates, and determining inhibition by observation of decreased enzymatic activity in the incubation with test sample in comparison to the control. In addition to using lysine, assays of the invention typically employ naturally-occuring isolated tRNA, total tRNA, synthetic substrates such as that prepared in the examples that follow, and fragments, variants, and mixtures thereof.

In addition to screening methods for enzymatic activity, the invention further provides screening methods for testing compounds, antibodies and the like, as inhibitors of LysRSI synthesis or stability, and adjunct compounds that enhance uptake, minimize side effects, and other properties required of inhibitors for certain medical and veterinary applications. The invention thus provides a way to identify compounds which can be used to inhibit LysRSI for therapeutic purposes as well as adjunct compounds. The unique sequence information is therefore useful for the development of therapeutically relevant compounds.

It is a further and important advantage of the invention that in all species observed to exhibit lysRSI activity do not exhibit the type II-type lysyl tRNA synthetase (LysRSII) activity common in most other species, including animals. Because the enzyme is essential for protein synthesis in the organisms, inhibitors and adjunct compound to be useful in screening methods of the invention to inhibit lysRSI activity can be used therapeutically to treat and control infectious agents exhibiting lysRSI activity Infectious agents include, but are not limited to, Borrelia burgdorferi, the causative agent of Lyme disease; other Borrelia that cause various types of relapsing fever; Treponema pallidum, the causative agent of syphilis, Leptospira and other spirochetes causing infectious hepatitis and various leptospiroses; as well as other pathogenic microorganisms having lysRSI and not lysRSII. Because of the fundamental difference in the lysyl-tRNA synthetase enzymes between pathogen and host, the pathogen can be specifically targeted and its replication, stopped without causing harm to the host For certain infections and pathological conditions, the invention therefore provides treatments that represent a marked improvement over broad-spectrum antibiotics that kill beneficial organism as well as pathogens.

As summarized above, the invention also provides valuable diagnostic screens. In a typical screen, a biological sample is obtained from an organism such as a mammal that is subject to infection by a microorganism expressing class I-type lysyl-tRNA synthetase. The presence of infection is determined by observation of class I-type lysyl-tRNA synthetase activity in the sample using any type of assay described above, or by observation of the polypeptide in the sample by standard means such as an ELISA, RIA, fluorochrome tag, and the like which typically employ an antibody or antibody fragment, including fusion phage as well as monoclonal antibodies, to detect the presence of the polypeptide.

Any type of biological sample suitable for an enzyme assay or polypeptide detection method may be employed in diagnostic screens according to the invention. Typical preferred samples are liquids. For disease detection in medical or veterinary patients or mammalian sampling of disease host reservoirs, plasma and serum may be employed, as can blood, saliva, tears, semen, amnionic fluid, and biopsy tissue. For disease detection in vector populations such as ticks in Borrelia screens, typical samples are ground arthropods or arthropod tissues. The invention thus provides new assays for Lyme disease, syphilis, and other pathological conditions caused by microorganisms expressing class I-type lysyl-tRNA synthetase.

Alternatively, polynucleotide sequences encompassed by the invention such as SEQ ID NOs 10 and 11 can be used in the design of probes for the detection of infection using RNA or DNA analysis. Identification of sequences encoding class I-type lysyl-tRNA synthetase in biological samples obtained from cultures, vectors, hosts, or patients indicates the presence of infection by the pathological microorganism.

EXAMPLES

The following examples are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard.

Example 1

To investigate the formation of Lys-tRNA in cell-free extracts prepared from M. maripaludis, (¹⁴C) lysine and homologous total TRNA were used as substrates (6). Amino acid analysis of the ¹⁴C labeled aminoacyl-tRNA produced in this reaction (7) indicated that lysine was directly acylated onto tRNA and therefore that Lys-tRNA was not the product of a tRNA-dependent amino acid transformation. This was confirmed by the observation that (¹⁴C)Lys-tRNA synthesis was inhibited by only one of the 20 canonical amino acids, lysine (FIG. 1). This is consistent with the presence of LysRS, since Lys-tRNA synthesis via a mischarged tRNA would result in a non-labeled amino acid other than lysine being able to inhibit lysylation of tRNA. On the basis of these observations LysRS from M. maripaludis was purified. By standard chromatographic procedures (8), a single 62-kD protein was isolated, which was purified to homogeneity as judged by both silver staining after denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and the appearance of a single symmetrical peak during size exclusion chromatography. The LysRS activity eluted as a single discrete fraction during all purification steps, indicating that M. maripaludis contains only a single LysRS activity. The enzyme could acylate unfractionated tRNA with lysine to the same degree as the M. maripaludis crude extract (FIG. 1), confirming that the purified protein is LysRS. In kinetic characterizations of our LysRS for the aminoacylation of M. maripaludis total tRNA with lysine (9), a K_(M) for lysine of 2.2 μM and a k_(cat) of 1.6 s⁻¹ were observed, values comparable to other LysRS proteins (10). Investigation of the species-specificity of Lys-tRNA formation in cell-free extracts showed reactivity between tRNA and RNA-free protein extracts prepared from E. coli, M. thermoautotrophicum, and M. maripaludis (11). The only exception was M. maripaludis tRNA, which is not a substrate for the E. coli enzyme, suggesting that it may lack 2-thiouridine and its derivatives which are required for the LysRS·tRNA^(Lys) interaction (12). These modified nucleotides have previously been detected at position 34 in all known tRNA^(Glu) and tRNA^(Lys) isoacceptors (13). In that these modifications are critical for recognition of the tRNA^(Lys) anticodon by LysRS (12), their possible absence suggests that tRNA recognition by the M. maripaludis enzyme is substantially different from known examples and may explain the inability of an E. coli extract to amino-acylate M. maripaludis tRNA with lysine.

A portion (22 amino acid residues) of the NH₂-terminal sequence of LysRS was determined by protein analysis and the sequence was used to clone lysS, the LysRS-encoding gene (SEQ ID NO: 10). This amino acid sequence is homologous to the NH₂-terminus of a predicted protein coding region (MJ0539) in the M. jannaschii genome and one in M. thermoautotrophicum, but finds no homology elsewhere in any public database. The coding sequence MJ0539 has been tentatively identified as a putative aminoacyl tRNA-synthetase (14). The NH₂-terminal sequence was used in conjunction with a conserved internal region in the M. jannaschii and M. thermoautotrophicum open reading frames (ORFs) to clone by the polymerase chain reaction (PCR) a 450 bp fragment of M. maripaludis lysS from genomic DNA. This fragment was ³²P-labeled and used to isolate a DNA fragment containing the 3′-terminal 1293 bp of the lysS gene from a genomic library. The remainder of lysS (306 bp) was cloned by PCR from genomic DNA with a 5′-primer based on the NH₂-terminal amino acid sequence of LysRS. After being sequenced, the two portions of lysS were used to derive a full-length clone (15). The lysS gene is 1599 bp in length and encodes a 61.3-kD protein, in good agreement with the molecular mass deduced for the native protein from SDS-PAGE (FIG. 2). To confirm the identity of the cloned gene as lysS, it was subcloned into pET15b, which allowed the overproduction and subsequent chromatographic purification of M. maripaludis LysRS as a hexahistidine (His₆) fusion protein (16). This purified His₆-LysRS was able to aminoacylate with lysine M. maripaludis total tRNA to the same extent and at the same rate as the native enzyme (FIG. 3).

The predicted amino acid sequence of M. maripaludis LysRS is homologous to putative proteins in Archaeoglobus fulgidus (14), M. thermoautotrophicum and M. jannaschii but otherwise appears to have little or no similarity to any known sequences outside of the Euryarchaeota, including that for LysRS from the Crenarchaeon Sulfolobus solfataricus (a normal class II enzyme). The only exception is a putative ORF in the Crenarchaeon Cenarchaeum sp. which shares approximately 28% amino acid identity with the euryarchaeal LysRS protein herein described, including extensive homology in the regions containing the HIGN (residues 37 to 40 of SEQ ID NO: 3) and KISSS (residues 280 to 285 of SEQ ID NO: 3) motifs. Thus, although this protein performs the enzymatic function of a conventional LysRS, the specific esterification of tRNA with lysine, catalysis is accomplished in the context of an amino acid landscape that lacks any sequences corresponding to motifs 1, 2 or 3 (17), which are found in known LysRS proteins now classified as class II aminoacyl-tRNA synthetases (24). Based on extensive similarities in their NH₂-terminal domains, LysRS, aspartyl- (AspRS), and asparaginyl-(AsnRS) tRNA synthetases were grouped as paralogous enzymes in subclass IIb (18). However, the euryarchaeal LysRS bears no similarity to AspRS (for the latter is normal) and AsnRS is absent, which leaves AspRS as the only member of this subclass. Thus there would appear to be considerably more evolutionary variation of the aminoacyl-tRNA synthetases than previously thought, a proposal supported by the apparent absence of a recognizable cysteinyl-tRNA synthetase in at least some of the Archaea (2, 3) and the existence in the M. jannaschii genome of an ORF (MJ1660) encoding an unidentified class II aminoacyl-tRNA synthetase similar to the α-subunit of phenylalanyl-tRNA synthetase (14).

The euryarchaeal LysRS proteins show variations of the HIGN (residues 37 to 40 of SEQ ID NO: 3) and KISSS (residues 280 to 285 of SEQ ID NO: 3) nucleotide binding motifs (FIG. 2) characteristic of class I aminoacyl-tRNA synthetases. The occurrence of such sequence motifs has been correlated by structural studies with the topology of the catalytic domain, class I aminoacyl-tRNA synthetases containing a Rossmann fold and class II an anti-parallel β sheet. The fact that euryarchaeal LysRS proteins show the defining motifs of class I aminoacyl-tRNA synthetases forces the unexpected conclusion that the catalytic domains of these enzymes are structurally unrelated to those of their bacterial, eukaryotic, and even certain crenarchaeal counter-parts which belong to class II (19). Phylogenetic analysis of an overall class I alignment (20) did not indicate any unequivocal specific relationship between euryarchaeal LysRS and any other class I aminoacyl-tRNA synthetase. Thus it is presently unclear whether the euryarchaeal-type LysRS was present in the last common ancestor or arose later through recruitment of another class I enzyme within the archaea. This is surprising because LysRS (like other aminoacyl-tRNA synthetases (20)) is conserved through evolution both in the other living kingdoms and in certain Crenarchaeota. To date, archaeal LysRS appears to represent the only known example of class switching among aminoacyl-tRNA synthetases and confirms the unexpected evolutionary origin of euryarchaeal LysRS. LysRS has so far been detected in both major branches of the Archaea; four euryarchaeotes (FIG. 3) and two crenarchaeotes, Cenarchaeum sp. and S. solfataricus. All show the unexpected class I LysRS except for S. solfataricus, which, like all other examples contains a class II LysRS (whether it also contains a class I LysRS is not known). This dichotomy is sufficiently complex and the number of archaeal examples are sufficiently small that speculation concerning LysRS evolution is not warranted at this stage. However, our data do cast doubt on previous evolutionary proposals based on the presumption of constancy of distribution of any given aminoacyl-tRNA synthetase. The observation that archaeal LysRS is a class I aminoacyl-tRNA synthetase also represents a functional demonstration of non-orthologous displacement (21) of a gene required for a core process in gene expression. It differs, for example, from archaeal histone-like proteins (22) and the recently discovered DNA topoisomerase VI of Sulfolobus shibatae (23), as these enzymes both have eukaryotic homologues of known function.

Example 2

Preliminary analysis of the genomic sequence of the bacterial pathogen Borrelia burgdorferi, the causative agent of Lyme disease, indicated the presence of an open reading frame encoding a protein with over 55% similarity at the amino acid level to the class I-type lysyl-tRNA synthetase of Methanococcus maripaludis (27) described in Example 1 above and shown in FIG. 2. In contrast, no coding region with significant similary to any class-II type lysyl-tRNA synthetase could be detected. In order to test the functionality of the protein encoded by this putative open reading frame (BblysS), the corresponding nucleotide sequence was amplified from a Borrelia total DNA preparation by the polymerase chain reaction. The product of this reaction was then subcloned into the vector pET15b as described in Example 1 above to facilitate heterologous expression of this open reading frame in E. coli. The E. coli strain BL21(DE3) was then transformed with this vector (pET15b-BblysS) and grown under standard conditions. Isopropyl β-D-thiogalactopyranoside-induced expression of the BblysS gene led to the production of a protein of the expected size (63.2 kD). This protein was then purified to electrophoretic homogeneity by standard chromatographic procedures. The protein displayed canonical lysyl-tRNA synthetase activity in vitro (FIG. 4), confirming that the BblysS gene encodes a functional homologue of Methanococcus maripaludis lysyl-tRNA synthetase. Thus, the lysyl-tRNA synthetases of Borrelia burgdorferi and Methanococcus maripaluis share a common origin. This result is surprising as all previously characterized bacterial and lysyl-tRNA synthetases are class II-type synthetases.

Example 3

In order to characterize the kinetic parameters of Borrelia burgdorferi lysyl-tRNA synthetase encoded in SEQ ID NO: 11, the nucelotide sequence encoding B. burgdorferi tRNALys1 (SEQ ID NO: 12) was amplified from a total DNA preparation by the polymerase chain reaction. The product was then subcloned into the vector pUC119 such that it was under transcriptional control of the T7 RNA polymerase promoter. The E. coli strain DH5α was then transformed with this vector (pUC119-Bb tRNALys1) and grown under standard conditions with ampicillin selection. The plasmid (pUC119-Bb tRNALys1) was then purified by standard procedures and subjected to enzymatic digestion by the restriction endonuclease BstN1. After purification by standard procedures, the cleaved plasmid DNA was used as a template for T7 RNA polymerase directed in vitro transcription. The RNA transcript produced by this reaction was then purified under denaturing conditions and refolded. This RNA transcript, which corresponds to an unmodified form of Borrelia burgdorferi tRNALys1, was then used for in vitro aminoacylation assays as described in Example 2 above. The rate of formation of Lys-tRNA was found to be the same with this synthetic substrate as previously observed for other class I-type lysyl-tRNA synthetases with tRNAs synthesized in vivo.

REFERENCES AND NOTES

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2. C. J. Bult et al., Science 273, 1058 (1996).

3. D. Smith et al., J. Bacteriol. (1997).

4. A. W. Curnow, M. Ibba, D. Söll, Nature 382, 589 (1996).

5. M. Ibba, A. W. Curnow, D. Söll, Trends Biochem. Sci. 22, 39 (1997).

6. Frozen M. maripaludis cells were resuspended in one volume of buffer A (25 mM Hepes, pH 7.2, 1 mM MgCl₂, 30 mM NaCl, 5 mM dithiothreitol (DTT), 4 mM 2-mercaptoethanol, 10% glycerol), sonicated, and centrifuged at 160,000 g for 3 hours. This extract (S160) was then exchanged into fresh buffer A and applied to a Q-Sepharose Fast Flow column (all chromatography columns were from Pharmacia). The major protein fraction was then eluted with buffer A containing 300 mM NaCl. Under these conditions, RNA remains bound to the column. The protein extract was then concentrated by ammonium sulfate precipitation, and resuspended and exchanged back into buffer A. M. maripaludis and M. thermoautotrophicum total tRNA were prepared by standard methods; E. coli total tRNA was from Boehringer Mannheim. Aminoacylation reactions were performed at 37° C. in 100 mM Hepes, pH 7.2, 50 mM KCl, 10 mM MgCl₂, 5 mM ATP, 5 mM DTT, BSA at 0.1 mg/ml, 20 μM (¹⁴C-U)lysine (317 μCi/μmol; NEN Dupont), and tRNA at 1 mg/ml.

7. For the analysis of the 3′-esterified amino acid, samples were removed from the aminoacylation reaction, and the attached amino acid was recovered as described (4). Samples were then applied to cellulose thin layer chromatography (TLC) plates which were developed in a mixture of methanol (6 parts):chloroform (6 parts):ammonium hydroxide (2 parts):water (1 part) (E. von Arx, and R. Neher, J. Chromatog. 12, 329 (1963)). ¹⁴C-labeled amino acids were visualized by phosphorimager and internal standards by ninhydrin staining.

8. Frozen cells (20g) were used to prepare an S160 extract as described above, from which the major protein fraction was then separated by ammonium sulfate precipitation. This fraction was dialyzed and applied to a Q-Sepharose Fast Flow column which was then developed with an NaCl gradient (0 to 300 mM). LysRS-containing fractions were pooled, dialyzed, and then fractionated by anion exchange chromatography with a Mono-Q column. Active fractions were again pooled and dialyzed and the pH was adjusted from 7.2 to 6, and the fractions applied to a Mono-S cation exchange column that was developed with an NaCl gradient (0 to 250 mM). The LysRS containing samples were pooled and concentrated before they were applied to a Superose 12 gel filtration column (buffer A, pH 7.2). The LysRS fractions from this final step were judged to be pure by silver staining after SDS-PAGE. Approximately 0.4 mg of LysRS were obtained by this procedure.

9. For the determination of kinetic parameters, LysRS was added to a final concentration of 5 nM and the lysine concentration was varied in the range 0.2 to 5 times the K_(M). Sampling and quantification were as described (K. -W. Hong et al., EMBO J. 15, 1983 (1996)).

10. W. Freist and D. H. Gauss, Biol. Chem. Hoppe Seyler 376, 451 (1995).

11. Significant lysylation represents at least 25% of the activity observed in the corresponding homologous system.

12. K. Tamura, H. Himeno, H. Ashahara, T. Hasegawa, M. Shimizu, Nucleic Acids Res. 20, 2335 (1992).

13. K. Watanabe et al., Nucleic Acids Res. 22, 79 (1994); G. R. Björk, in tRNA: Structure, Biosynthesis, and Function, D. Söll and U. L. RajBhandary, Eds. (ASM Press, Washington, D.C., 1995), p.165; S. Cusack, A. Yaremchuk, M. Tukalo, EMBO J. 15, 6321 (1996).

14. A. R. Kerlavage, et al. The complete genome sequence of the hyperthermophilic, sulphate-reducing aracheon Archaeoglobus fulgidus. The Institute for Genomic Research, Rockville, Md. 20850. Retrieved from the Internet: <URL: http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=gaf>. Sequence published in Nature 390:364-370 (1997); erratum in Nature 394: 101 (1998).

15. An internal fragment of the lysS gene was cloned from M. maripaludis genomic DNA by2 PCR with the primers CCNTG(TC)CCNGA(AG)GGNTG(TC)TG(TC)GA(AG)AG (KRS1, SEQ ID NO: 13) and GCNCCNG(CA)NGCNGCAG(TG)(AG)TC(TC)TTNCC (KRS2, SEQ ID NO: 14). This probe was then ³²P-labeled and hybridized to a M. maripaludis genomic Zap Express (Stratagene) λ library. This allowed isolation of the 3′ end of lysS (over 80% of the gene). The remaining 5′ region was cloned by PCR from genomic DNA using as primers KRS2 and KRS3 (ATGCA(TC)TGGGCNGA(TC)GCNAC, SEQ ID NO: 15). Both strands of these fragments were sequenced by dye labeling, and no differences were found in the overlapping regions. The complete lysS ORF was then generated by overlap-extension PCR using the two cloned fragments as templates with the primers KRS3 and KRS4 (GTATCCTCTTCAAACTCGTTAGGAC, SEQ ID NO: 16), which is complementary to a sequence 3′ from the stop codon of lysS.

16. For expression in E. coli, M. maripaludis lysS was subcloned into pET15b (Invitrogen) and then used to transform the strain BL21 (DE3) (W. F. Studier, A. H. Rosenberg, J. J. Dunn, J. W. Dudendorf, Methods Enzymol. 85, 60 (1990)). This transformation allowed the production of His₆-LysRS, which was subsequently purified by nickel-affinity chromatography (Qiagen) followed by gel filtration with a Superose 12 column (8).

17. G. Eriani, M. Delarue, O. Poch, J. Gangloff, D. Moras, Nature 347, 203 (1990).

18. S. Cusack, Nature Struct. Biol. 2, 824 (1995).

19. S. Onesti, A. D. Miller, P. Brick, Structure 3, 163 (1995).

20. A phylogenetic tree was constructed by the maximum parsimony method (J. R. Brown and W. F. Doolittle, Proc. Natl. Acad. Sci. U.S.A. 92, 2441 (1995); G. M. Nagel and R. F. Doolittle, J. Mol. Evol. 40, 487 (1995)). Amino acid sequences for the HIGN (residues 37 to 40 of SEQ ID NO: 3) and KISSS (residues 280 to 285 of SEQ ID NO: 3) motifs and flanking residues were aligned with CLUSTAL W (J. D. Thompson, D. G. Higgins, T. J. Gibson, Nucleic Acids Res. 22, 4673 (1994)) and all insertions and deletions then removed. A maximum parsimony tree was derived with the use of the programs SEQBOOT, PROTPARS, and CONSENSE from the PHYLIP 3.5c package (J. Felsenstein, Phylogeny Inference Package. Dept. Genet., Univ. Washington, Seattle (1993)). This gave rise to a phylogenetic tree with low replicate frequencies for more than 50% of the nodes.

21. A. R. Mushegian and E. V. Koonin, Proc. Natl. Acad. Sci. U.S.A 93, 10258 (1996).

22. R. A. Grayling, K. Sandman, J. N. Reeve, FEMS Microbiol. Revs. 18, 203 (1996); C. A. Ouzounis and N. C. Kyrpides, J. Mol. Evol. 42, 234 (1996).

23. A. Bergerat et al., Nature 386, 414 (1997).

24. Sequence alignment of motifs 1, 2, and 3 (18, 19) from class II LysRS enzymes against the most similar regions in euryarachaeal LysRS enzymes were determined. Class II and euryarchaeal sequences were separately aligned with CLUSTAL W and the aligned class II motifs (1, 2 or 3) used individually to search for similar regions in the euryarchaeal enzymes. The class II LysRS sequences from Cricetulus longicaudatus (GenBank accession number: Z31711), Homo sapiens (D31890), Caenorhabditis elegans (U41105), Saccharomyces cerevisiae cytoplasm (X56259), Lycopersicon esculentum (X94451), E. coli lysS (U28375), E. coli lysU (X16542), Haemophilus influenzae (P43825), Acinetobacter calcoaceticus (Z46863), Bacillus subtilis (P37477), Staphylococcus aureus (L36472), Synechocystis sp. (D90906), Campylobacter jejuni (M63448), Thermus thermophilus (P41255), Mycoplasma genitalium (P47382), Mycoplasma pneumoniae (AE000055), Mycoplasma fermentans (U50825), Mycoplasma hominis (P46191), S. solfataricus (Y08257) and S. cerevisiae mitochondria (X57360).

25. The sequence of the M. maripaludis lysS gene has been deposited in GenBank, accession no. AF009824.

26. Ibba, M., Morgan, S., Curnow, A. W., Pridmore, D. R., Vothknecht, U. C., Gardner, W., Lin, W., Woese, C. R., and Söll, D., Science 278, 1119 (1997).

27. Ibba, M., Bono, J. L., Rosa, P. A., and Söll, D., Proc. Natl. Acad. Sci. USA 94, 14383 (1997).

The papers cited in the above section and elsewhere in the application are expressly incorporated herein in their entireties by reference.

The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

The U.S. government has certain rights in the invention as it was made with partial support from N.I.G.M.S. grant number GM22854.

16 1 494 PRT Pyrococcus furiosus lysyl t-RNA synthetase 1 Met Val His Trp Ala Asp Tyr Met Ala Glu Lys Ile Ile Lys Glu 5 10 15 Arg Gly Glu Lys Glu Glu Tyr Val Val Glu Ser Gly Ile Thr Pro 20 25 30 Ser Gly Tyr Val His Val Gly Asn Phe Arg Glu Leu Phe Thr Ala 35 40 45 Tyr Ile Val Gly His Ala Leu Arg Asp Arg Gly Tyr Asn Val Arg 50 55 60 His Ile His Met Trp Asp Asp Tyr Asp Arg Phe Arg Lys Val Pro 65 70 75 Lys Asn Val Pro Gln Glu Trp Glu Glu Tyr Leu Gly Met Pro Val 80 85 90 Ser Glu Val Pro Asp Pro Trp Gly Cys His Asp Ser Tyr Ala Glu 95 100 105 His Phe Met Gly Leu Phe Glu Glu Glu Val Ala Lys Leu Glu Met 110 115 120 Asp Val Glu Phe Leu Arg Ala Ser Glu Leu Tyr Lys Lys Gly Glu 125 130 135 Tyr Ala Glu Glu Ile Arg Lys Ala Phe Glu Ala Lys Gly Lys Ile 140 145 150 Met Ala Ile Leu Asn Lys Tyr Arg Glu Val Ala Lys Gln Pro Pro 155 160 165 Leu Pro Glu Asn Trp Trp Pro Ala Met Val Tyr Cys Pro Glu His 170 175 180 Arg Lys Glu Ser Glu Ile Ile Asp Trp Asp Gly Glu Trp Gly Val 185 190 195 Lys Tyr Arg Cys Pro Glu Gly His Glu Gly Trp Thr Asp Ile Arg 200 205 210 Asp Gly Asn Val Lys Leu Arg Trp Arg Val Asp Trp Pro Met Arg 215 220 225 Trp Ala His Phe Gly Val Asp Phe Glu Pro Ala Gly Lys Asp His 230 235 240 Leu Ala Ala Gly Ser Ser Tyr Asp Thr Gly Lys Glu Ile Ile Arg 245 250 255 Glu Val Tyr Gly Lys Glu Ala Pro Leu Thr Leu Met Tyr Glu Phe 260 265 270 Val Gly Ile Lys Gly Gln Lys Gly Lys Met Ser Gly Ser Lys Gly 275 280 285 Asn Val Ile Leu Leu Ser Asp Leu Tyr Glu Val Leu Glu Pro Gly 290 295 300 Leu Val Arg Phe Ile Tyr Ala Lys His Arg Pro Asn Lys Glu Ile 305 310 315 Arg Ile Asp Leu Gly Leu Gly Leu Leu Asn Leu Tyr Asp Glu Phe 320 325 330 Asp Arg Val Glu Arg Ile Tyr Phe Gly Ile Glu Lys Gly Lys Gly 335 340 345 Asp Glu Glu Glu Leu Lys Arg Thr Tyr Glu Leu Ser Val Pro Lys 350 355 360 Lys Pro Lys Arg Leu Val Ala Gln Ala Pro Phe Arg Phe Leu Ala 365 370 375 Val Leu Val Gln Leu Pro His Leu Ser Ile Glu Asp Ile Ile Phe 380 385 390 Thr Leu Val Lys Gln Gly His Val Pro Glu Asn Leu Thr Gln Glu 395 400 405 Asp Ile Asp Arg Ile Lys Leu Arg Ile Lys Leu Ala Lys Asn Trp 410 415 420 Val Glu Lys Tyr Ala Pro Glu Glu Val Lys Phe Lys Ile Leu Ser 425 430 435 Val Pro Gly Val Ser Glu Val Asp Pro Thr Ile Arg Glu Ala Met 440 445 450 Leu Glu Val Ala Glu Trp Leu Glu Ser His Glu Asp Phe Ala Val 455 460 465 Asp Glu Leu Asn Asn Ile Leu Phe Glu Val Ala Lys Lys Arg Asn 470 475 480 Ile Pro Ser Lys Val Trp Phe Ser His Cys Thr Asn Tyr Ser 485 490 2 523 PRT Pyrococcus horikoshii lysyl t-RNA synthetase 2 Met Val His Trp Ala Asp Tyr Ile Ala Asp Lys Ile Ile Arg Glu 5 10 15 Arg Gly Glu Lys Glu Lys Tyr Val Val Glu Ser Gly Ile Thr Pro 20 25 30 Ser Gly Tyr Val His Val Gly Asn Phe Arg Glu Leu Phe Thr Ala 35 40 45 Tyr Ile Val Gly His Ala Leu Arg Asp Lys Gly Tyr Glu Val Arg 50 55 60 His Ile His Met Trp Asp Asp Tyr Asp Arg Phe Arg Lys Val Pro 65 70 75 Arg Asn Val Pro Gln Glu Trp Lys Asp Tyr Leu Gly Met Pro Ile 80 85 90 Ser Glu Val Pro Asp Pro Trp Gly Cys His Glu Ser Tyr Ala Glu 95 100 105 His Phe Met Arg Lys Phe Glu Glu Glu Val Glu Lys Leu Gly Ile 110 115 120 Glu Val Asp Phe Leu Tyr Ala Ser Glu Leu Tyr Lys Arg Gly Glu 125 130 135 Tyr Ser Glu Glu Ile Arg Leu Ala Phe Glu Lys Arg Asp Lys Ile 140 145 150 Met Glu Ile Leu Asn Lys Tyr Arg Glu Ile Ala Lys Gln Pro Pro 155 160 165 Leu Pro Glu Asn Trp Trp Pro Ala Met Val Tyr Cys Pro Glu His 170 175 180 Arg Arg Glu Ala Glu Ile Ile Glu Trp Asp Gly Gly Trp Lys Val 185 190 195 Lys Tyr Lys Cys Pro Glu Gly His Glu Gly Trp Val Asp Ile Arg 200 205 210 Ser Gly Asn Val Lys Leu Arg Trp Arg Val Asp Trp Pro Met Arg 215 220 225 Trp Ser His Phe Gly Val Asp Phe Glu Pro Ala Gly Lys Asp His 230 235 240 Leu Val Ala Gly Ser Ser Tyr Asp Thr Gly Lys Glu Ile Ile Lys 245 250 255 Glu Val Tyr Gly Lys Glu Ala Pro Leu Ser Leu Met Tyr Glu Phe 260 265 270 Val Gly Ile Lys Gly Gln Lys Gly Lys Met Ser Gly Ser Lys Gly 275 280 285 Asn Val Ile Leu Leu Ser Asp Leu Tyr Glu Val Leu Glu Pro Gly 290 295 300 Leu Val Arg Phe Ile Tyr Ala Arg His Arg Pro Asn Lys Glu Ile 305 310 315 Lys Ile Asp Leu Gly Leu Gly Ile Leu Asn Leu Tyr Asp Glu Phe 320 325 330 Asp Lys Val Glu Arg Ile Tyr Phe Gly Val Glu Gly Gly Lys Gly 335 340 345 Asp Asp Glu Glu Leu Arg Arg Thr Tyr Glu Leu Ser Met Pro Lys 350 355 360 Lys Pro Glu Arg Leu Val Ala Gln Ala Pro Phe Arg Phe Leu Ala 365 370 375 Val Leu Val Gln Leu Pro His Leu Thr Glu Glu Asp Ile Ile Asn 380 385 390 Val Leu Ile Lys Gln Gly His Ile Pro Arg Asp Leu Ser Lys Glu 395 400 405 Asp Val Glu Arg Val Lys Leu Arg Ile Asn Leu Ala Arg Asn Trp 410 415 420 Val Lys Lys Tyr Ala Pro Glu Asp Val Lys Phe Ser Ile Leu Glu 425 430 435 Lys Pro Pro Glu Val Glu Val Ser Glu Asp Val Arg Glu Ala Met 440 445 450 Asn Glu Val Ala Glu Trp Leu Glu Asn His Glu Glu Phe Ser Val 455 460 465 Glu Glu Phe Asn Asn Ile Leu Phe Glu Val Ala Lys Arg Arg Gly 470 475 480 Ile Ser Ser Arg Glu Trp Phe Ser Thr Leu Tyr Arg Leu Phe Ile 485 490 495 Gly Lys Glu Arg Gly Pro Arg Leu Ala Ser Phe Leu Ala Ser Leu 500 505 510 Asp Arg Ser Phe Val Ile Lys Arg Leu Arg Leu Glu Gly 515 520 3 521 PRT Borrelia burgdorferi lysyl t-RNA synthetase construct expressed in Example 3 3 Met Lys Thr Ala His Trp Ala Asp Phe Tyr Ala Glu Lys Ile Lys 5 10 15 Lys Glu Lys Gly Pro Lys Asn Leu Tyr Thr Val Ala Ser Gly Ile 20 25 30 Thr Pro Ser Gly Thr Val His Ile Gly Asn Phe Arg Glu Val Ile 35 40 45 Ser Val Asp Leu Val Ala Arg Ala Leu Arg Asp Ser Gly Ser Lys 50 55 60 Val Arg Phe Ile Tyr Ser Trp Asp Asn Tyr Asp Val Phe Arg Lys 65 70 75 Val Pro Lys Asn Met Pro Glu Gln Glu Leu Leu Thr Thr Tyr Leu 80 85 90 Arg Gln Ala Ile Thr Arg Val Pro Asp Thr Arg Ser His Lys Thr 95 100 105 Ser Tyr Ala Arg Ala Asn Glu Ile Glu Phe Glu Lys Tyr Leu Pro 110 115 120 Val Val Gly Ile Asn Pro Glu Phe Ile Asp Gln Ser Lys Gln Tyr 125 130 135 Thr Ser Asn Ala Tyr Ala Ser Gln Ile Lys Phe Ala Leu Asp His 140 145 150 Lys Lys Glu Leu Ser Glu Ala Leu Asn Glu Tyr Arg Thr Ser Lys 155 160 165 Leu Glu Glu Asn Trp Tyr Pro Ile Ser Val Phe Cys Thr Lys Cys 170 175 180 Asn Arg Asp Thr Thr Thr Val Asn Asn Tyr Asp Asn His Tyr Ser 185 190 195 Val Glu Tyr Ser Cys Glu Cys Gly Asn Gln Glu Ser Leu Asp Ile 200 205 210 Arg Thr Thr Trp Ala Ile Lys Leu Pro Trp Arg Ile Asp Trp Pro 215 220 225 Met Arg Trp Lys Tyr Glu Lys Val Asp Phe Glu Pro Ala Gly Lys 230 235 240 Asp His His Ser Ser Gly Gly Ser Phe Asp Thr Ser Lys Asn Ile 245 250 255 Val Lys Ile Phe Gln Gly Ser Pro Pro Val Thr Phe Gln Tyr Asp 260 265 270 Phe Ile Ser Ile Lys Gly Arg Gly Gly Lys Ile Ser Ser Ser Ser 275 280 285 Gly Asp Val Ile Ser Leu Lys Asp Val Leu Glu Val Tyr Thr Pro 290 295 300 Glu Val Thr Arg Phe Leu Phe Ala Ala Thr Lys Pro Asn Thr Glu 305 310 315 Phe Ser Ile Ser Phe Asp Leu Asp Val Ile Lys Ile Tyr Glu Asp 320 325 330 Tyr Asp Lys Phe Glu Arg Ile Tyr Tyr Gly Val Glu Asp Val Lys 335 340 345 Glu Glu Lys Lys Arg Ala Phe Lys Arg Ile Tyr Glu Leu Ser Gln 350 355 360 Pro Tyr Met Pro Ser Lys Arg Ile Pro Tyr Gln Val Gly Phe Arg 365 370 375 His Leu Ser Val Ile Ser Gln Ile Phe Glu Asn Asn Ile Asn Lys 380 385 390 Ile Leu Asn Tyr Leu Lys Asn Val Gln Glu Asp Gln Lys Asp Lys 395 400 405 Leu Ile Asn Lys Ile Asn Cys Ala Ile Asn Trp Ile Arg Asp Phe 410 415 420 Ala Pro Glu Asp Phe Lys Phe Ser Leu Arg Ser Lys Phe Asp Asn 425 430 435 Met Glu Ile Leu Glu Glu Asn Ser Lys Lys Ala Ile Asn Glu Leu 440 445 450 Leu Asp Phe Leu Lys Lys Asn Phe Glu Val Ala Thr Glu Gln Asp 455 460 465 Ile Gln Asn Glu Ile Tyr Lys Ile Ser Arg Glu Asn Asn Ile Glu 470 475 480 Pro Ala Leu Phe Phe Lys Gln Ile Tyr Lys Ile Leu Ile Asp Lys 485 490 495 Glu Lys Gly Pro Lys Leu Ala Gly Phe Ile Lys Ile Ile Gly Ile 500 505 510 Asp Arg Phe Glu Lys Ile Thr Ser Lys Tyr Val 515 520 4 528 PRT Treponema pallidum lysyl t-RNA synthetase 4 Met Ser Ile Cys Glu Lys Ser Leu His Trp Ala Asp Lys Val Ala 5 10 15 His Lys Ile Ile Lys Glu Arg Ala Asp Cys Asp Gln Tyr Thr Cys 20 25 30 Ala Ser Gly Ile Thr Pro Ser Gly Thr Val His Ile Gly Asn Phe 35 40 45 Arg Glu Ile Ile Ser Val Asp Leu Val Val Arg Ala Leu Arg Asp 50 55 60 Gln Gly Lys Ser Val Arg Phe Val His Ser Trp Asp Asp Tyr Asp 65 70 75 Val Phe Arg Arg Ile Pro Asp Asn Val Pro Ala Gln Asp Glu Leu 80 85 90 Lys Gln Tyr Ile Arg Met Pro Ile Thr Ser Val Pro Asp Pro Phe 95 100 105 Gln Gln Glu Asp Ser Tyr Ala Arg His His Glu Arg Glu Ile Glu 110 115 120 Ser Ala Leu Pro Glu Val Gly Ile Tyr Pro Glu Tyr Val Tyr Gln 125 130 135 Ser Lys Gln Tyr Gln Ala Gly Val Tyr Ala Gln Glu Ile Lys Ile 140 145 150 Ala Leu Asp Asn Arg His Arg Ile Gln Ala Ile Leu Asn Glu Tyr 155 160 165 Arg Asp Glu Gln His Lys Ile Ser Gly Thr Tyr Trp Pro Val Ser 170 175 180 Val Phe Cys Thr Ala Cys His Lys Asp Cys Thr Thr Val Asp Ala 185 190 195 Trp Asp Ser His Trp Cys Leu Gln Tyr His Cys Glu Cys Gly His 200 205 210 Gly Glu Gln Val Asp Leu Arg Gln Thr Ser Ala Val Lys Leu Ser 215 220 225 Trp Arg Val Asp Trp Ala Met Arg Trp Ser Lys Glu His Val Val 230 235 240 Phe Glu Pro Ala Gly Lys Asp His His Ser Gln Gly Gly Ser Phe 245 250 255 Asp Thr Ala Arg Leu Ile Ser Asp His Ile Tyr His Trp Pro Ala 260 265 270 Pro Val Ser Phe Arg Tyr Asp Phe Ile Gly Leu Lys Gly Met Pro 275 280 285 Gly Lys Met Ser Ser Ser Ala Gly Lys Val Val Gly Leu Arg Asp 290 295 300 Val Leu Glu Val Tyr Gln Pro Glu Val Leu Arg Tyr Leu Phe Val 305 310 315 Ser Thr Arg Pro Asn Thr Glu Phe Ser Ile Ser Phe Asp Leu Asp 320 325 330 Val Leu Lys Ile Tyr Glu Asp Tyr Asp Lys Ser Glu Arg Val Ala 335 340 345 Trp Gly Ile His Ala Ala Lys Ser Glu His Glu Phe Met Arg His 350 355 360 Lys Arg Ile Tyr Glu Leu Ser Gln Val Arg Gly Met Pro Pro Cys 365 370 375 Ile Ser Tyr Gln Val Pro Phe Arg His Val Cys Asn Ile Leu Gln 380 385 390 Ile Asn Ser Gly Asp Ile Ser Ala Val Leu Ala Phe Phe Ser Asp 395 400 405 Ile His Lys Asp Gln Ile Glu Arg Phe Val Arg Arg Cys Gln Cys 410 415 420 Ala Trp Asn Trp Ile Arg Asp Ala Gly Ala Pro Asp Asp Phe Lys 425 430 435 Phe Thr Leu Lys Glu Asp Gly Val Arg Val Pro Leu Ser Ala Glu 440 445 450 Ile Thr Glu Ala Leu Arg Leu Ile Arg Asp Thr Leu Val Pro Arg 455 460 465 Thr Asp Val Leu Ser Glu Lys Glu Leu Ser Ala Glu Leu Tyr Ala 470 475 480 Val Ala Arg Gln Ile Pro Val Gly Ser Lys Glu Leu Phe Thr Ala 485 490 495 Leu Tyr Gln Val Leu Ile Gly Lys Asn Gln Gly Pro Arg Leu Ala 500 505 510 Gly Phe Met Lys Val Ile Gly Thr Gln Arg Leu His Arg Met Leu 515 520 525 Ser Val Tyr 5 534 PRT Methanococcus jannaschii lysyl t-RNA synthetase 5 Leu Arg Glu Ile Met His Trp Ala Asp Val Ile Ala Glu Lys Leu 5 10 15 Ile Glu Glu Arg Lys Ala Asp Lys Tyr Ile Val Ala Ser Gly Ile 20 25 30 Thr Pro Ser Gly His Ile His Val Gly Asn Ala Arg Glu Thr Leu 35 40 45 Thr Ala Asp Ala Ile Tyr Lys Gly Leu Ile Asn Lys Gly Val Glu 50 55 60 Ala Glu Leu Ile Phe Ile Ala Asp Thr Tyr Asp Pro Leu Arg Lys 65 70 75 Leu Tyr Pro Phe Leu Pro Lys Glu Phe Glu Gln Tyr Ile Gly Met 80 85 90 Pro Leu Ser Glu Ile Pro Cys Pro Glu Gly Cys Cys Glu Ser Tyr 95 100 105 Ala Glu His Phe Leu Arg Pro Tyr Leu Glu Ser Leu Asp Asp Leu 110 115 120 Gly Val Glu Leu Thr Thr Tyr Arg Ala Asp Glu Asn Tyr Lys Lys 125 130 135 Gly Leu Tyr Asp Glu Lys Ile Lys Ile Ala Leu Asp Asn Arg Glu 140 145 150 Lys Ile Met Glu Ile Leu Asn Lys Phe Arg Ala Asn Pro Leu Pro 155 160 165 Asp Asp Trp Trp Pro Ile Asn Ile Val Cys Glu Asn Cys Gly Lys 170 175 180 Leu Lys Thr Lys Val Ile Lys Tyr Asp Ser Glu Lys Glu Glu Ile 185 190 195 Thr Tyr Arg Cys Glu Ile Cys Gly Phe Glu Asn Thr Val Lys Pro 200 205 210 Tyr Lys Gly Arg Ala Lys Leu Pro Trp Arg Val Asp Trp Pro Ala 215 220 225 Arg Trp Ser Ile Phe Asn Val Thr Ile Glu Pro Met Gly Lys Asp 230 235 240 His Ala Ala Ala Gly Gly Ser Tyr Asp Thr Gly Val Leu Ile Ala 245 250 255 Lys Glu Ile Tyr Asn Tyr Ile Pro Pro Lys Lys Val Val Tyr Glu 260 265 270 Trp Ile Gln Leu Lys Val Gly Asp Lys Ala Ile Pro Met Ser Ser 275 280 285 Ser Lys Gly Val Val Phe Ala Val Lys Asp Trp Thr Asn Ile Ala 290 295 300 His Pro Glu Ile Leu Arg Phe Leu Leu Leu Arg Ser Lys Pro Thr 305 310 315 Lys His Ile Asp Phe Asp Leu Lys Lys Ile Pro Asp Leu Val Asp 320 325 330 Glu Tyr Asp Arg Leu Glu Asp Phe Tyr Phe Asn Asn Lys Asp Lys 335 340 345 Asp Glu Leu Ser Glu Glu Glu Gln Glu Lys Ile Arg Ile Tyr Glu 350 355 360 Leu Ser Thr Pro Lys Ile Pro Glu Thr Lys Pro Phe Val Ile Pro 365 370 375 Tyr Arg Phe Cys Ser Ile Ile Ala Gln Leu Thr Tyr Asp Glu Glu 380 385 390 Lys Glu Asp Ile Asn Met Glu Arg Val Phe Glu Ile Leu Arg Arg 395 400 405 Asn Asn Tyr Ser Ile Asp Asp Ile Asp Glu Phe Ser Met Lys Lys 410 415 420 Leu Lys Asp Arg Leu Leu Met Ala Arg Asn Trp Ala Leu Lys Tyr 425 430 435 Gly Glu Lys Leu Val Ile Ile Ser Glu Asp Glu Ala Lys Glu Ile 440 445 450 Tyr Glu Lys Leu Lys Asp Lys Gln Lys Glu Trp Ile Lys Tyr Phe 455 460 465 Ala Glu Lys Leu Lys Thr Ala Glu Phe Asp Ala Leu Asn Leu His 470 475 480 Glu Leu Ile Tyr Gln Thr Ala Lys Glu Leu Gly Leu Asn Pro Arg 485 490 495 Asp Ala Phe Gln Ala Ser Tyr Met Ile Leu Leu Gly Lys Lys Tyr 500 505 510 Gly Pro Lys Leu Gly Ala Phe Leu Ala Thr Leu Gly Lys Asp Phe 515 520 525 Val Ile Arg Arg Tyr Ser Leu Phe Glu 530 6 533 PRT Methanococcus maripaludis lysyl t-RNA synthetase construct expressed in Example 1 6 Met His Trp Ala Asp Ala Thr Ser Glu Lys Ile Met Lys Lys Arg 5 10 15 Asn Ala Glu Glu Tyr Val Val Ser Ser Gly Ile Thr Pro Ser Gly 20 25 30 His Ile His Ile Gly Asn Ala Arg Glu Thr Leu Thr Ala Asp Ala 35 40 45 Val Tyr Lys Gly Met Leu Lys Lys Gly Ala Glu Ala Lys Leu Ile 50 55 60 Phe Ile Ala Asp Asp Tyr Asp Pro Leu Arg Lys Leu Tyr Pro Phe 65 70 75 Leu Pro Lys Glu Phe Glu Lys Tyr Ile Gly Met Pro Leu Ser Glu 80 85 90 Ile Pro Cys Pro Gln Gly Cys Cys Lys Ser Tyr Ala Asp His Phe 95 100 105 Leu Met Pro Phe Leu Asn Ser Leu Glu Asp Leu Gly Val Glu Ile 110 115 120 Thr Thr His Arg Ala Asn Glu Cys Tyr Lys Ala Gly Met Tyr Asn 125 130 135 Glu Ala Ile Ile Thr Ala Leu Glu Asn Arg Leu Lys Ile Lys Glu 140 145 150 Leu Leu Asp Ser Tyr Arg Lys Glu Pro Leu Ala Asp Asn Trp Tyr 155 160 165 Pro Leu Asn Val Val Cys Glu Lys Cys Gly Lys Met His Glu Thr 170 175 180 Lys Val Thr Ser Tyr Asn Ser Glu Asp Lys Thr Ile Thr Tyr Val 185 190 195 Cys Lys Cys Gly Phe Glu Asn Thr Val Gln Pro Phe Asn Gly Ile 200 205 210 Gly Lys Leu Pro Trp Arg Val Asp Trp Pro Ala Arg Trp Asn Ile 215 220 225 Phe Gly Val Thr Ala Glu Pro Met Gly Lys Asp His Ala Ala Ser 230 235 240 Gly Gly Ser Tyr Asp Thr Gly Ile Lys Ile Ala Arg Gln Ile Phe 245 250 255 Asn Tyr Gln Ala Pro Glu Lys Met Val Tyr Glu Trp Ile Gln Leu 260 265 270 Lys Ile Gly Asp Lys Ala Met Pro Met Ser Ser Ser Ser Gly Val 275 280 285 Val Phe Ala Val Lys Asp Trp Thr Glu Ile Cys His Pro Glu Val 290 295 300 Leu Arg Phe Leu Ile Leu Lys Gly Lys Pro Thr Lys His Ile Asp 305 310 315 Phe Asp Leu Lys Ala Ile Ser Asn Leu Val Asp Asp Tyr Asp Glu 320 325 330 Leu Glu Arg Lys Tyr Phe Glu Leu Ile Glu Lys Gln Lys Thr Glu 335 340 345 Glu Leu Asn Asp Asn Glu Asn Glu Lys Ile Ser Leu Tyr Glu Leu 350 355 360 Val Thr Pro Lys Ile Pro Glu Arg Leu Pro Leu Gln Val Ala Tyr 365 370 375 Arg Phe Cys Ser Ile Ile Ala Gln Ile Ala Leu Asp Lys Glu Thr 380 385 390 Gln Lys Ile Asp Met Glu Arg Val Phe Asp Ile Leu Gly Arg Asn 395 400 405 Gly Tyr Asn Pro Ala Glu Phe Ser Glu Tyr Asp Lys Ser Arg Leu 410 415 420 Glu Lys Arg Leu Tyr Met Ser Lys Lys Trp Ala Ser Asp Tyr Gly 425 430 435 Glu Asn Leu Glu Ile Asn Asp Phe Glu Gln Ala Lys Glu Gln Tyr 440 445 450 Glu Thr Leu Ser Glu Glu Gln Lys Ala Trp Leu Lys Ala Phe Ser 455 460 465 Lys Glu Val Glu Asn Ile Glu Ile Asp Ala Asn Thr Ile His Glu 470 475 480 Leu Met Tyr Glu Thr Ala Thr Lys Leu Asn Leu Ala Pro Lys Glu 485 490 495 Ala Phe Val Ala Ser Tyr Lys Ile Leu Leu Gly Lys Asn Tyr Gly 500 505 510 Pro Lys Leu Gly Ser Phe Leu Ala Ser Leu Lys Lys Glu Phe Val 515 520 525 Ile Gly Arg Phe Asn Leu Thr Glu 530 7 493 PRT Archaeolglobus fulgidus lysyl t-RNA synthetase 7 Met His Trp Ala Asp Val Ile Ala Ala Asp Leu Leu Lys Arg Ser 5 10 15 Asn Ser His Arg Ile Ala Thr Gly Ile Ser Pro Ser Gly His Ile 20 25 30 His Leu Gly Asn Leu Arg Glu Met Val Thr Ala Asp Ala Ile Arg 35 40 45 Arg Ala Leu Leu Asp Ala Gly Gly Glu Ala Lys Ile Val Tyr Ile 50 55 60 Ala Asp Asp Phe Asp Pro Leu Arg Arg Arg Tyr Pro Phe Leu Pro 65 70 75 Glu Glu Tyr Glu Asn Tyr Val Gly Met Pro Leu Cys Lys Ile Pro 80 85 90 Asp Pro Glu Gly Cys His Asp Ser Tyr Ser Glu His Phe Leu Gln 95 100 105 Pro Phe Leu Glu Ser Leu Glu Ile Leu Gly Ile Pro Val Glu Val 110 115 120 Arg Arg Ala Tyr Gln Met Tyr Ser Glu Gly Leu Tyr Glu Asn Asn 125 130 135 Thr Arg Ile Ala Leu Lys Arg Arg Asp Glu Ile Ala Arg Ile Ile 140 145 150 Ala Glu Val Thr Gly Arg Glu Leu Glu Glu Arg Trp Tyr Pro Phe 155 160 165 Met Pro Leu Cys Glu Asn Cys Gly Arg Ile Asn Ser Thr Arg Val 170 175 180 Thr Ser Phe Asp Glu Asn Trp Ile Tyr Tyr Glu Cys Asp Cys Gly 185 190 195 His Ser Gly Arg Val Gly Tyr Val Gly Gly Gly Lys Leu Thr Trp 200 205 210 Arg Val Asp Trp Ala Ala Arg Trp Gln Ile Leu Ser Ile Thr Cys 215 220 225 Glu Pro Phe Gly Lys Asp His Ala Ala Ala Gly Gly Ser Tyr Asp 230 235 240 Thr Gly Val Arg Ile Ala Arg Glu Ile Phe Asp Tyr Glu Pro Pro 245 250 255 Tyr Pro Val Pro Tyr Glu Trp Ile His Leu Lys Gly Lys Gly Ala 260 265 270 Met Lys Ser Ser Lys Gly Ile Val Leu Pro Val Arg Glu Met Val 275 280 285 Glu Val Ile Pro Pro Glu Ile Val Arg Tyr Ile Thr Ile Arg Val 290 295 300 Lys Pro Glu Arg His Ile Glu Phe Asp Pro Gly Leu Gly Leu Leu 305 310 315 Asp Leu Val Glu Glu Phe Glu Glu Lys Phe Lys Glu Lys Asp Arg 320 325 330 Ser Val Glu Leu Ser Leu Val Gly Glu Val Val Tyr Ser Asp Val 335 340 345 Pro Phe Arg His Leu Ile Val Val Gly Gln Ile Ala Asn Trp Asp 350 355 360 Leu Glu Lys Ala Leu Glu Ile Ile Glu Arg Thr Gly Tyr Thr Val 365 370 375 Asp Asp Val Thr Arg Arg Asp Val Glu Arg Arg Leu Lys Tyr Ala 380 385 390 Arg Lys Trp Leu Glu Lys Tyr Ala Pro Asp Asn Ile Lys Phe Glu 395 400 405 Ile Pro Glu Lys Val Thr Ala Glu Phe Ser Glu Glu Glu Lys Lys 410 415 420 Phe Leu Arg Ala Tyr Ala Glu Arg Leu Arg Ser Asp Met Lys Pro 425 430 435 Glu Glu Ile His Thr Leu Val Tyr Asp Val Ser Lys Glu Val Gly 440 445 450 Ile Lys Ser Ser Lys Ala Phe Gln Ala Ile Tyr Lys Ala Ile Leu 455 460 465 Gly Lys Thr Tyr Gly Pro Arg Val Gly Tyr Phe Ile Lys Ser Leu 470 475 480 Gly Val Glu Trp Val Arg Glu Arg Ile Lys Ala Ala Leu 485 490 8 513 PRT Methanobacterium thermoautotrophicum lysyl t-RNA synthetase 8 Leu Lys Glu Arg Asp Val Glu Glu His Val Val Ala Ser Gly Thr 5 10 15 Ser Ile Ser Gly Ser Ile His Ile Gly Asn Ser Cys Asp Val Phe 20 25 30 Ile Ala Ser Ser Ile Ala Lys Ser Leu Lys Lys Asp Gly Phe Lys 35 40 45 Ser Arg Thr Val Trp Ile Ala Asp Asp His Asp Pro Leu Arg Lys 50 55 60 Val Pro Tyr Pro Leu Pro Glu Ser Tyr Glu Lys Tyr Leu Gly Val 65 70 75 Pro Tyr Ser Met Ile Pro Cys Pro Glu Gly Cys Cys Glu Ser Phe 80 85 90 Val Glu His Phe Gln Arg Pro Phe Leu Glu Ala Leu Glu Arg Phe 95 100 105 Arg Ile Gly Val Glu His Tyr Ser Gly Ala Arg Met Tyr Thr Glu 110 115 120 Gly Leu Tyr Asn Asp Tyr Ile Arg Thr Ser Leu Glu Arg Ala Pro 125 130 135 Glu Ile Arg Glu Ile Phe Asn Arg Phe Arg Asp Arg Pro Leu Arg 140 145 150 Asp Asp Trp Leu Pro Tyr Asn Pro Ile Cys Glu Lys Cys Gly Arg 155 160 165 Val Asn Thr Thr Glu Ala Tyr Asp Phe Ser Gly Asp Thr Val Arg 170 175 180 Tyr Arg Cys Glu Cys Gly Phe Asp Gly Glu Met Asp Ile Lys Ser 185 190 195 Gly Leu Gly Lys Leu Thr Trp Arg Val Glu Trp Ala Ala Arg Trp 200 205 210 Lys Ile Leu Gly Val Thr Cys Glu Pro Phe Gly Lys Asp His Ala 215 220 225 Ala Ser Gly Gly Ser Tyr Asp Val Ser Ser Ile Ile Ser Glu Glu 230 235 240 Ile Phe Asp Tyr Pro Ala Pro Tyr Pro Val Pro Tyr Glu Trp Ile 245 250 255 Thr Leu Arg Gly Glu Ala Met Ser Lys Ser Arg Gly Val Phe Phe 260 265 270 Thr Pro Gly Gln Trp Leu Glu Ile Gly Pro Pro Glu Ser Leu Asn 275 280 285 Tyr Phe Ile Phe Arg Ser Lys Pro Met Lys His Lys Asp Phe Asn 290 295 300 Pro Asp Met Pro Phe Leu Asp Leu Met Asp Gln Phe Asp Arg Thr 305 310 315 Glu Arg Ile Tyr Tyr Gly Met Glu Asp Ala Ala Ser Glu Lys Glu 320 325 330 Glu Gln Lys Leu Arg Asn Ile Tyr Arg Val Ser Met Ile Glu Glu 335 340 345 Phe Asp Leu Pro Leu Arg Pro Ser Tyr Arg Phe Met Thr Val Ala 350 355 360 Cys Gln Ile Ala Gly Asp Asp Pro Glu Arg Leu Tyr Asp Ile Leu 365 370 375 Arg Arg Asn Ser Gln Leu Pro Glu Glu Leu Met Asp Leu Glu Leu 380 385 390 Asp Gln Leu Thr Asp Lys Gln Leu Glu Gln Leu Asn Glu Arg Ile 395 400 405 Glu Asn Val Lys Asn Trp Leu Arg Leu Tyr Ala Pro Glu Phe Val 410 415 420 Lys Phe Gln Val Gln Glu Glu Leu Pro Asp Val Glu Leu Ser Glu 425 430 435 Pro Gln Leu Lys Phe Leu Gln Asp Val Ala Asp Leu Met Glu Ser 440 445 450 Arg Glu Met Ser Ala Glu Glu Leu His Asp Glu Met Tyr Ser Ile 455 460 465 Leu Arg Arg His Gly Leu Lys Pro Gln Lys Ala Phe Gln Ala Ile 470 475 480 Tyr Arg Val Leu Ile Gly Lys Lys Met Gly Pro Arg Ala Ala Ser 485 490 495 Phe Leu Leu Ser Leu Glu Arg Asp Phe Val Ile Arg Arg Leu Arg 500 505 510 Leu Glu Ala 9 564 PRT Rhodobacter capsulatus lysyl t-RNA synthetase 9 Leu Gly Leu Pro Gln Glu Leu Leu Thr Phe Gln Ala Pro Phe Asn 5 10 15 Arg Ser Gly Ala Arg Arg Met Gly Arg Pro Asp Glu Asp Leu Thr 20 25 30 Met Thr Thr Leu Arg Asp Ala Ala Met Asn Ser Lys Ala Trp Pro 35 40 45 Phe Glu Glu Ala Arg Arg Val Leu Lys Arg Tyr Glu Lys Ala Pro 50 55 60 Pro Lys Lys Gly His Val Leu Phe Glu Thr Gly Tyr Gly Pro Ser 65 70 75 Gly Leu Pro His Ile Gly Thr Phe Gly Glu Val Ala Arg Thr Thr 80 85 90 Met Ile Arg Arg Ala Phe Glu Ile Ile Ser Asp Ile Pro Thr Arg 95 100 105 Leu Leu Cys Phe Ser Asp Asp Met Asp Gly Met Arg Lys Val Pro 110 115 120 Glu Asn Val Pro Gln Gln Glu Leu Leu Tyr Ala Asn Ile Gln Lys 125 130 135 Pro Leu Thr Ala Val Pro Asp Pro Phe Gly Glu Phe Asp Ser Phe 140 145 150 Gly Asn His Asn Asn Ala Met Leu Arg Arg Phe Leu Asp Thr Phe 155 160 165 Gly Phe Glu Tyr Glu Phe Ala Ser Ala Thr Asp Tyr Tyr Lys Ser 170 175 180 Gly Lys Phe Asp Glu Met Leu Met Leu Cys Ala Glu Arg Tyr Asp 185 190 195 Ala Ile Met Ala Ile Met Leu Lys Ser Leu Arg Glu Glu Arg Gln 200 205 210 Gln Thr Tyr Ser Cys Phe Leu Pro Ile His Pro Glu Thr Gly Arg 215 220 225 Val Leu Tyr Val Pro Met Lys His Val Asp Ala Lys Asn Gly Leu 230 235 240 Ile Thr Phe Asp Asp Glu Asp Gly Arg Glu Trp Thr Leu Pro Val 245 250 255 Thr Gly Gly Lys Val Lys Leu Gln Trp Lys Pro Asp Phe Gly Met 260 265 270 Arg Trp Ala Ala Leu Asp Val Asp Phe Glu Met Tyr Gly Lys Asp 275 280 285 His Ser Thr Asn Thr Pro Ile Tyr Asp Gly Ile Cys Glu Val Leu 290 295 300 Gly Gly Arg Lys Pro Glu His Phe Thr Tyr Glu Leu Phe Leu Asp 305 310 315 Asp Gln Gly Gln Lys Ile Ser Lys Ser Lys Gly Asn Gly Leu Thr 320 325 330 Ile Asp Glu Trp Leu Ser Tyr Ala Ala Thr Glu Ser Leu Ser Tyr 335 340 345 Phe Met Tyr Gln Lys Pro Lys Thr Ala Lys Arg Leu Trp Trp Asp 350 355 360 Val Ile Pro Lys Ala Val Asp Glu Tyr His Gln Gln Leu Arg Ala 365 370 375 Tyr Pro Thr Gln Pro Val Glu Lys Gln Ile Asp Asn Pro Val Trp 380 385 390 His Ile His Gly Gly Gln Pro Pro Glu Ser Asn Leu Val Val Pro 395 400 405 Phe Ala Met Leu Leu Asn Leu Ala Ser Val Ala Gly Ala Ser Asp 410 415 420 Lys Ala Gly Leu Trp Gly Phe Ile Lys Arg Tyr Ala Pro Glu Ala 425 430 435 Thr Pro Glu Thr His Pro Asp Leu Asp Ala Ala Ala Gly Phe Ala 440 445 450 Val Arg Tyr Phe His Asp Phe Val Ala Pro Thr Arg Thr Phe Arg 455 460 465 Leu Pro Ser Asp Lys Glu Arg Ala Ala Met Glu Asp Leu Leu Ala 470 475 480 Arg Leu Lys Ala Leu Pro Ala Thr Asp Leu His Leu Gln Phe Glu 485 490 495 Asp Pro Ser Glu Gly Leu Gln Thr Ile Val Phe Ala Val Gly Thr 500 505 510 Glu His Gly Phe Glu Pro Leu Arg Asp Trp Phe Thr Ala Leu Tyr 515 520 525 Glu Val Leu Leu Gly Gln Ser Gln Gly Pro Arg Phe Gly Gly Phe 530 535 540 Ile Ala Leu Tyr Gly Val Ser Glu Thr Ile Ala Leu Met Glu Thr 545 550 555 Ala Leu Ala Gly Gly Leu Ile Lys Ala 560 10 1602 DNA Methanococcus maripaludis lysyl t-RNA synthetase clone described in Example 1 10 atgcattggg cagatgctac atcggaaaaa atcatgaaaa aaagaaatgc 50 agaggaatac gtggtatcaa gtggaattac tccatctggt cacattcaca 100 ttgggaatgc aagggaaaca ttaactgcag atgcagttta caagggcatg 150 ctaaaaaaag gtgcagaagc taaattaatt tttattgcgg acgattatga 200 tcctttaaga aaattatatc catttttacc aaaagaattt gaaaaataca 250 taggaatgcc gttaagtgaa attccatgcc cacaaggttg ctgtaaaagc 300 tatgcagatc attttttaat gccgttttta aacagtcttg aagatttagg 350 cgttgaaatt actacccata gggcaaatga atgctataaa gctggaatgt 400 acaacgaagc aattataact gcacttgaaa accggttaaa aatcaaagaa 450 cttctcgatt cttaccgaaa agaaccactt gctgataatt ggtatccttt 500 aaacgttgta tgtgaaaaat gcggtaaaat gcacgaaaca aaagttacca 550 gttacaattc tgaagacaaa acaataactt acgtttgtaa atgcggattt 600 gaaaacacgg ttcaaccatt taacggcatt ggaaagcttc cgtggagagt 650 tgactggcct gcaagatgga atatatttgg agttactgct gaaccgatgg 700 gaaaagacca cgcagcgtca ggcggttcat acgatacagg aattaagatt 750 gcaagacaga ttttcaacta ccaagcacca gaaaaaatgg tttacgagtg 800 gattcagtta aaaatcgggg ataaggcaat gccaatgtct tcttcatcag 850 gagttgtatt tgcagtaaaa gactggactg aaatctgcca ccctgaagtt 900 ttaagatttt taattttaaa aggaaaacca acaaaacaca tagattttga 950 tttaaaagca atttcaaatt tagttgacga ttacgacgaa cttgaaagaa 1000 aatactttga attaatcgaa aaacaaaaaa ctgaagaatt aaacgacaac 1050 gaaaatgaaa aaataagttt atatgaactt gttacaccaa aaatacctga 1100 aagattacca ttacaagttg catacaggtt ctgttcaatt attgctcaga 1150 ttgcactcga taaagaaacc caaaaaattg atatggaaag agttttcgac 1200 attttgggaa gaaatggata caatccagca gaattttcag aatacgacaa 1250 atcaaggctt gaaaaaaggc tttatatgtc taaaaaatgg gcatccgact 1300 acggtgaaaa tttagaaata aatgattttg agcaagcaaa agagcagtac 1350 gaaacactct cagaagagca aaaagcatgg ttaaaagcat tttccaaaga 1400 agtagaaaat atcgaaattg atgcgaacac gattcacgaa ttgatgtatg 1450 aaactgcaac aaaattaaat cttgcaccaa aagaagcgtt tgttgcatca 1500 tacaaaatac ttctcggtaa aaactacggg ccaaagttag gaagcttttt 1550 agcatccctt aaaaaagagt tcgtaattgg aagatttaat ttaacagaat 1600 aa 1602 11 1563 DNA Borrelia burgdorferi lysyl t-RNA synthetase clone described in Example 3 11 gtgaaaacag cacactgggc agatttttac gcagaaaaaa taaaaaaaga 50 aaaaggtcca aaaaacttat acacagtagc atcgggaatt actccatctg 100 gaactgtgca cattggcaat tttagagaag ttatttcggt agaccttgta 150 gcaagagcac taagagactc tggatcaaaa gtaaggttta tttattcttg 200 ggataattac gacgtatttc gaaaagttcc caaaaatatg ccagaacaag 250 aacttcttac aacttattta agacaagcaa taacaagggt ccctgacaca 300 agaagccaca aaacaagtta tgcaagggct aatgaaattg aatttgaaaa 350 atatctgcct gtagttggga tcaatcctga attcatcgac caaagcaaac 400 aatataccag caacgcttat gcaagccaaa taaaatttgc acttgatcat 450 aaaaaagaac tgtctgaagc attaaacgaa tacagaacct caaagcttga 500 agaaaattgg tatccaatca gtgtattttg tacaaaatgc aatagagaca 550 caacaactgt aaataattat gacaatcatt actctgttga gtattcatgt 600 gaatgtggaa atcaagaatc tctagacata agaaccacat gggccattaa 650 acttccttgg agaatagatt ggcctatgag atggaaatat gaaaaagttg 700 actttgagcc tgcaggaaaa gaccaccaca gcagtggcgg cagttttgat 750 acatctaaaa atattgtaaa aatttttcaa ggtagccctc ctgtaacatt 800 tcaatatgac tttatttcaa taaaaggacg tggtggaaaa atatcctcct 850 catcgggaga tgtcatatcg ctcaaagatg ttcttgaggt ctatacaccc 900 gaagtcacaa ggtttttatt tgctgctact aaaccaaata ctgaattttc 950 aatctcattt gatcttgatg taattaaaat atacgaagat tacgacaaat 1000 ttgagagaat ctactatgga gtagaagatg taaaagaaga aaaaaaaaga 1050 gcatttaaaa gaatttacga actatctcaa ccatacatgc caagcaaaag 1100 aatcccttat caggtcggat tcagacattt aagtgtaatc agtcaaatat 1150 ttgaaaataa tataaataaa attttaaatt acttgaaaaa cgttcaagaa 1200 gatcaaaaag acaaactaat aaataaaata aattgcgcaa ttaattggat 1250 aagagatttt gcacccgaag atttcaaatt ttcattaaga tctaaatttg 1300 ataatatgga aatactagaa gaaaatagca aaaaagcaat taatgaactt 1350 ttggattttt taaagaaaaa ttttgaagtt gccacagaac aagacattca 1400 aaacgaaata tataaaattt caagagaaaa taatatagaa cctgctttat 1450 tttttaaaca aatttataaa attttaattg acaaagaaaa agggcccaaa 1500 ttagctggat ttatcaaaat aattggtatt gatcgctttg aaaagattac 1550 aagcaaatac gtt 1563 12 73 DNA Borrelia burgdorferi t-RNA synthetase Lys1 sequence described in Example 3 12 gggctcatag ctcaggtggt agagcagcgc ccttttaagg cgtttgtcgt 50 aggttcgagt cctactgagc tca 73 13 31 DNA artificial sequence KRS1 primer employed in note 15 n = A or C, or G or T, or other 13 ccntgtcccn gaagggntgt ctgtcgaaga g 31 14 30 DNA artificial sequence KRS2 primer employed in note 15; n = A or C, or G or T, or other 14 gcnccngcan gcngcagtga gtctcttncc 30 15 22 DNA artificial sequence KRS3 primer employed in note 15; n = A or C, or G or T, or other 15 atgcatctgg gcngatcgcn ac 22 16 25 DNA artificial sequence KRS4 primer employed in note 15 16 gtatcctctt caaactcgtt aggac 25 

What is claimed is:
 1. An isolated polypeptide comprising a lysyl-tRNA synthetase having at least about 85% sequence identity with SEQ ID NO: 3 or SEQ ID NO:
 6. 2. A polypeptide according to claim 1 which bas at least about 90% sequence identity with SEQ ID NO:
 3. 3. A polypeptide according to claim 2 which is SEQ ID NO:
 3. 4. A polypeptide according to claim 1 which is a Borrelia burgdorferi class I-type lysyl tRNA synthetase.
 5. A polypeptide according to claim 1 which has at least about 90% sequence identity with SEQ ID NO:
 6. 6. A polypeptide according to claim 1 which is SEQ ID NO:
 6. 7. A method for screening for inhibitors of Borrelia burgdorferi class I-type lysyl-tRNA synthetase enzyme inhibition using test molecules comprising (a) incubating a test molecule with an enzymatic polypeptide corresponding to SEQ ID NO: 3 in a mixture containing both lysine and tRNA for a time under conditions sufficient to observe acylation of the same tRNA with lysine in a control incubation of an enzymatic polypeptide corresponding to SEQ ID NO: 3 with lysine and the same tRNA under the same conditions, and (b) determining inhibition by observation of decreased acylation of the tRNA with lysine in the incubation with test molecule in comparison with a control.
 8. A method according to claim 7 wherein the tRNA is naturally-occurring isolated B. burgdorferi tRNA.
 9. A method according to claim 7 wherein the tRNA is synthetic tRNA encoded by SEQ ID NO:
 12. 10. A method according to claim 7 wherein the tRNA is E. coli tRNA. 